The Study Of HSV-tk Gene Expression And Antitumor Activity Mediated By Tetracycline-regulatable System In Vitro

Some research has shown that HSV-tk/GCV systems exhibits not only direct cytotoxicity but also a bystander effect. Furthermore, it has been demonstrated HSV-tk/GCV can induce tumor-specific immunity that leads to rejection implant untransduced tumor cells in vivo. However, liver failure caused by HSV-tk/GCV systems was discovered by several studys. Therefore, the HSV-tk gene expression must be accurately regulated in target cells.Tetracycline regulated system is one of the most versatile tools for controlling gene expression available today. The system is relatively non-toxic to cells in culture and the induction of target gene expression is rapid and reversible. The system has been widely utilized due to these favorable features.The aim of this study is to construct tetracycline-regulatable HSV-tk gene system with double plasmids vector and a single plasmid vector for regulating expression and tumor killed activity in HeLa cells and HCT-8 cells.1. To construct the vector forβ-galactosidase gene expression regulation with tetracycline-regulated gene expression system, and to controlβ-galactosidase gene expression in hepG2 cell with the vector.To design primers for expanding two tetracycline operator (TetO2) gene according to the gene nucleotide sequences of TetO2 gene and pcDNA3.1 vector. To amplify TetO2 gene by PCR with pcDNA3.1 plasmid as template. The TetO2 PCR products was cloned into pcDNA3.1 and the vector was named as pcDNA3.1-TetO2. The pcDNA3.1-Teto2 with TetO2 PCR products sequence was analyzed by ABI3130 sequencing analysis. Thenβ-galactosidase gene (β-gal ) was cloned into pcDNA3.1-TetO2, the vector was named as pcDNA3.1-TetO2-β-gal. The pcDNA6/TR plasmid vector with tetracycline repressor(TetR) gene were transfected into HepG2 cells by Lipotap, and the stable transfection cells were screened by blasticidin. TR gene expression was detected by RT-PCR in HepG2 cells with and without pcDNA6/TR plasmid transfection. The pcDNA3.1-TetO2-β-gal plasmid vector were transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection, and after 3 to 4 days, these transfected gene cells deal with doxycline(4μg/ml). After 48 h,β-gal gene expression was detected withβ-galactosidase cell staining.Results: The pcDNA3.1-TetO2 sequencing analysis results showed that a cassette for a cytomegalovirus-type 2 tetracycline operator (TetO2)-TetO2 promoter in pcDNA3.1-TetO2. The double digestion result of pcDNA3.1-TetO2-β-gal plasmid vector demonstrate thatβ-gal gene was successfully cloned into pcDNA3.1-TetO2 vector .The RT-PCR result of TR gene showed TR gene could be expressed in HepG2 cells with pcDNA6/TR plasmid stable transfection and TR gene expression have not been shown in HepG2 cells without pcDNA6/TR plasmid transfection.β-gal gene can be expressed in HepG2 cells without pcDNA6/TR plasmid transfection, but can be expressed in HepG2 cells with pcDNA6/TR plasmid transfection. However,β-gal gene expression can be induced with doxycline in HepG2 cells with pcDNA6/TR plasmid transfection. The tetracycline-regulated gene expression system vector for regulatingβ-galactosidase gene expression was successful constructed and the vector system can controlβ-galactosidase gene expression in hepG2 cells.2.To construct the vector for HSV-tk gene expression regulation with tetracycline-regulated gene expression system, and to control HSV-tk gene expression and tumorcided in HeLa cell with the vector.The HSV-tk gene was cloned into pcDNA3.1-TetO2 vector and vector that can regulate HSV-tk gene expression was made. The pcDNA3.1-TetO2-HSV-tk plasmid vector were transfected into HepG2 cells with and without pcDNA6/TR plasmid transfection, and after 3 to 4 days, medium supplemented was replaced with G418 (800μg/ml) and blasticidin for screening gene stable transfection . To deal with these transfected gene cells with different concentrations of doxycline. And the HSV-tk mRNA levels were measured by reverse transcription polymerase chain reaction (RT-PCR). Cells were cultured for 72 h in the presence of different concentrations (0.1μg/mL, 1μg/mL, 10μg/mL, 50μg/mL, 100μg/mL,) of GCV and Dox(4μg/ml), The cell viability was assayed by MTT after 3 days.The results showed HSV-tk gene was successfully cloned into pcDNA3.1-TetO2 vector by double digestion. The HSV-tk gene can be expressed in HeLa cells without pcDNA6/TR plasmid transfection, but can not be expressed in HeLa cells with pcDNA6/TR plasmid transfection. However, HSV-tk gene expression can be induced with doxycline in HeLa cells with pcDNA6/TR plasmid transfection. No evident cytotoxicity was noted on the HeLa cells with TR pcDNA3.1-TetO2- HSV-tk gene transfection were treated with GCV concentrations of up to 100μg/ml. Without adding deoxycycline but the HeLa cells with gene transfection transfection with adding deoxycycline has higher sensitive to GCV, and the concentrations of GCV that produced a 50% decrease in cell viability was about 10μg/ml.3. To construct a single tetracycline-regulatable plasmid vector base on the double tetracycline-regulatable plasmid vector system and validate the capability for regulating HSV-tk gene expression and tumor killed activity in HeLa cells and HCT-8 cells.The HSV-tk and IRES gene were cloned into pcDNA3.1 -TetO2 to generate pcDNA3.1–TetO2-HSV-TK-IRES plasmid. The TR gene was cloned into plasmid pcDNA3.1–TetO2- HSV-tk-IRES to generate pcDNA3.1–TetO2-HSV- tk-IRES-TR plasmid. The HSV-tk and TR gene were linked by IRES so that both genes can be co- translated separately from a single, bicistronic mRNA .Plasmids pcDNA3.1-TetO2-HSV-tk-IRES-TR were stable transfected into HeLa cells and HCT-8 cells by liposome. To deal with these transfected gene cells with different concentration doxycline. And the HSV-tk mRNA levels were measured by reverse transcription polymerase chain reaction (RT-PCR). Cells were cultured for 72 h in the presence of different concentrations (0.1μg/mL, 1μg/mL, 10μg/mL ,50μg/mL, 100μg/mL,) of GCV and Dox(4μg/ml). The cell viability was assayed by MTT assay.The results showed the HSV-tk gene was lower expression in HeLa and HCT-8 transfecting with pcDNA3.1-CMV-TetO2-HSV-tk-IRES-TR vector. The expression of HSV-tk was notably increased in HeLa and HCT-8 transfecting with pcDNA3.1- -TetO2-HSV-tk-IRES-TR vector when the different concentration of dox was added. Without dox,the HeLa cells with gene transfection transfection was lower sensitive to GCV, and the concentrations of GCV that produced a 50% decrease in cell viability was about 50μg/mL,the HCT-8 cells with gene transfection transfection was lower sensitive to GCV, and the concentrations of GCV that produced a 50% decrease in cell viability was about 100μg/mL. But with dox,the HeLa cells with gene transfection was higher sensitive to GCV, and the concentrations of GCV that produced a 50% decrease in cell viability was about 5μg/mL ,the HCT-8 cells with gene transfection was higher sensitive to GCV, and the concentrations of GCV that produced a 50% decrease in cell viability was about 10μg/mL.The empirical study obtain as following progression:(1) The vector forβ-galactosidase gene expression regulation with tetracycline- regulated gene expression system was successfully constructed, and can controlβ-galactosidase gene expression in hepG2 cell with the vector.(2) The vector for HSV-TK gene expression regulation with tetracycline- regulated gene expression system was constructed and can control HSV-TK gene expression and tumorcided in HeLa cell with the vector.(3) A single tetracycline-regulatable plasmid vector base on the double tetracycline-regulatable plasmid vector system was constructed and validated the capability for regulating HSV-tk gene expression and tumor killed activity in HeLa cells and HCT-8 cells.In conclusion,the tetracycline-regulatable plasmid vector for regulating HSV-tk gene expression has been constructed. The HSV-tk gene expression and sensitivity of GCV can be regulated with Dox by the vector in HeLa cells and Hct-8 cells.